Callus browning is a significant problem that hinders plant tissue regeneration in Paeonia ostii “Fengdan” by causing cell death and inhibiting growth. However, the molecular mechanism underlying callus browning in P. ostii remains unclear. In this study, we investigated the metabolites and potential regulatory genes involved in callus browning of P. ostii using metabolomic and transcriptomic analyses. We found a significant accumulation of phenolic compounds in the browned callus, represented by flavonoid compounds. Notably, the accumulations of luteotin and disomentin were higher in browning calli compared to non-browning calli. Transcriptomic analysis identified that candidate genes associated with flavonoid biosynthesis, including flavonoid 3-hydroxylase (PoF3H) and flavone synthase II (PoFNSII), were highly expressed in the browned callus of P. ostii “Fengdan”. Weighted gene co-expression network analysis (WGCNA) further highlighted that polyphenol oxidase (PoPPO) which encoded polyphenol oxidase, together with flavonoid biosynthesis-related genes such as flavanone 3-hydroxylase (PoF3H) and flavonone Synthase II (PoFNSII), as well as cellular totipotency-related genes wuschel-related homeobox 4 (PoWOX4), were involved in callus browning. Based on these findings, we proposed the molecular mechanism by which flavonoid accumulation, polyphenol oxidation, and cellular totipotency pathways contribute to callus browning in P. ostii. Our study provides new insights into the molecular mechanism underlying callus browning and offers the foundations to facilitate the establishment of an efficient plant tissue regeneration system in P. ostii.

MYBs constitute the second largest transcription factor (TF) superfamily in flowering plants with substantial structural and functional diversity, which have been brought into focus because they affect flower colors by regulating anthocyanin biosynthesis. Up to now, the genomic data of several Chrysanthemum species have been released, which provides us with abundant genomic resources for revealing the evolution of the MYB gene family in Chrysanthemum species. In the present study, comparative analyses of the MYB gene family in six representative species, including C. lavandulifolium, C. seticuspe, C. ×morifolium, Helianthus annuus, Lactuca sativa, and Arabidopsis thaliana, were performed. A total of 1104 MYBs, which were classified into four subfamilies and 35 lineages, were identified in the three Chrysanthemum species (C. lavandulifolium, C. seticuspe, and C. ×morifolium). We found that whole-genome duplication and tandem duplication are the main duplication mechanisms that drove the occurrence of duplicates in CmMYBs (particularly in the R2R3-MYB subfamily) during the evolution of the cultivated chrysanthemums. Sequence structure and selective pressure analyses of the MYB gene family revealed that some of R2R3-MYBs were subjected to positive selection, which are mostly located on the distal telomere segments of the chromosomes and contain motifs 7 and 8. In addition, the gene expression analysis of CmMYBs in different organs and at various capitulum developmental stages of C. ×morifolium indicated that CmMYBS2, CmMYB96, and CmMYB109 might be the negative regulators for anthocyanin biosynthesis. Our results provide the phylogenetic context for research on the genetic and functional evolution of the MYB gene family in Chrysanthemum species and deepen our understanding of the regulatory mechanism of MYB TFs on the flower color of C. ×morifolium.

Chrysanthemum × morifolium has great ornamental and economic value on account of its exquisite capitulum. However, previous studies have mainly focused on the corolla morphology of the capitulum. Such an approach cannot explain the variable inflorescence architecture of the chrysanthemum. Previous research from our group has shown that NO APICAL MERISTEM (ClNAM) is likely to function as a hub gene in capitulum architecture in the early development stage. In the present study, ClNAM was used to investigate the function of these boundary genes in the capitulum architecture of Chrysanthemum lavandulifolium, a closely related species of C. × morifolium in the genus. Modification of ClNAM in C. lavandulifolium resulted in an advanced initiation of the floral primordium at the capitulum. As a result, the receptacle morphology was altered and the number of florets decreased. The ray floret corolla was shortened, but the disc floret was elongated. The number of capitula increased significantly, arranged in more densely compounded corymbose synflorescences. The yeast and luciferase reporter system revealed that ClAP1, ClRCD2, and ClLBD18 target and activate ClNAM. Subsequently, ClNAM targets and activates ClCUC2a/c, which regulates the initiation of floral and inflorescence in C. lavandulifolium. ClNAM was also targeted and cleaved by cla-miR164 in this process. In conclusion, this study established a boundary gene regulatory network with cla-miR164-ClNAM as the hub. This network not only influences the architecture of capitulum, but also affects compound corymbose synflorescences of the C. lavandulifolium. These results provide new insights into the mechanisms regulating inflorescence architecture in chrysanthemum.

Helleborus orientalis L. is a valuable winter-flowering and understory landscape plant, but its application and breeding are hindered by poor heat tolerance and the lack of a robust germplasm evaluation system. In this study, 51 Helleborus L. hybrid individuals obtained through manual open pollination were evaluated using coefficient of variation (CV), Shannon–Weaver diversity index (H′), correlation analysis, principal component analysis (PCA), and cluster analysis to assess genetic diversity and ornamental value based on 17 phenotypic traits. The results showed rich phenotypic diversity among the hybrids. Quantitative traits showed CV ranging from 9.48% to 37.99% and H′ between 0.77 and 1.51, with flower count and leaf length being the most variable. Qualitative traits had H′ values from 0.52 to 1.55, with sepal color showing the highest diversity. Significant correlations were detected among heat tolerance, pest resistance, leaf and petiole length, as well as plant and flower form. PCA extracted six principal components accounting for 74.50% of cumulative variance. Cluster analysis classified the 51 germplasms into five groups. Using the AHP model, a comprehensive evaluation system was established, and 13 elite individuals were selected for variety rights application and characterization. This study provides a reference for establishing DUS test guidelines and advancing breeding and utilization of Helleborus L.

Paeonia lactiflora Pall. (herbaceous peony) is a high-value ornamental and medicinal plant in China with considerable market potential. However, conventional propagation methods are limited by low multiplication rates and long production cycles, making it difficult to meet the demand for large-scale planting materials. As a key approach for rapid propagation, tissue culture techniques for P. lactiflora have achieved significant progress in recent years. This review summarizes advances in the tissue culture system for P. lactiflora over the past decade, focusing on major in vitro regeneration pathways (organogenesis and somatic embryogenesis) and crucial technical stages, including explant selection and culture environment optimization. Distinct from previous reviews that only introduce partial technical aspects of P. lactiflora tissue culture, this review comprehensively outlines the overall tissue culture system, analyses the current species-specific bottlenecks (browning, vitrification, rooting and acclimatization) with their underlying causes and proposes targeted strategies. Furthermore, future development trends are prospected by integrating emerging research directions, including molecular regulatory mechanisms and eco-adaptive breeding. This review aims to provide a theoretical foundation and technical support for obtaining propagule for commercial plantations and achieving multi-functional utilization of P. lactiflora.